Method for repairing neurodegeneration

ABSTRACT

The present invention provides for treating neurodegeneration caused by nerve compression syndrome or entrapment neuropathy comprising administering human acidic fibroblast growth factor (aFGF), fibrinogen, aprotinin and divalent calcium ions to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/856,086, filed on Aug. 13, 2010, which claims the benefit of U.S.Provisional Application No. 61/233,987, filed on Aug. 14, 2009. Thecontents of both prior applications are hereby incorporated by referencein their entirety.

BACKGROUND

Nerve compressive syndrome, also known as entrapment neuropathy, is amedical condition caused by direct pressure, structural changes in thenerve or adjacent tissues or mechanically injury on a single nerve. Itssymptoms include pain, tingling, numbness, and muscle weakness. Thesymptoms affect just one particular part of the body, depending on whichnerve is affected. Although a well known example is compression of themedian nerve at the wrist (carpal tunnel syndrome), other nerves, suchas the ulnar nerve at the wrist or the elbow and the spinal nerve rootsat the vertebral foramen, are vulnerable. Long term effect of nervecompression may lead to neuron degeneration and the loss of neuralfunction.

Decompression surgery is a regular therapeutic method to remove thecompressive source, such as bone or cartilage, to release a pressure andmitigate the neuropathic pain. After surgery, the symptoms may resolvecompletely, but if the compression was sufficiently severe or prolonged,the nerve may not recover fully and some symptoms may persist. Moreover,the functional recovery of the patients after the surgery will needlonger time to be achieved. In most cases, the degeneration of nervefiber cannot be repaired with the decompression surgery.

Therefore, there is still a need on discovering a method for functionalimprovement or recovery of degenerated nerves in the early stage ofentrapment neuropathy or after the decompression surgery.

SUMMARY

The invention unexpectedly discovers that acid fibroblast growth factor(aFGF) plays a critical role in neural regeneration. When administeringaFGF to a subject suffering nerve compressive syndrome, it was observedfunctional recovery of the degenerative or injured nerve. In comparisonwith the traditional decompression surgery, injection aFGF is moreeffective and safer treatment.

Accordingly, the invention is to provide a method for treatingneurodegeneration caused by nerve compression syndrome or entrapmentneuropathy comprising administering human acidic fibroblast growthfactor (aFGF) to a subject in need thereof.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

In the drawings:

FIG. 1 is a diagram showing the grasping power test performed by therats having compression surgery administered saline or human aFGF byforamen magnum injection. * indicates the significant different betweenaFGF treatment group and control group (p<0.05). # indicates thesignificant different between pre-treatment and post-treatment of aFGF(p<0.05).

DETAILED DESCRIPTION

As used herein, the article “a” or “an” means one or more than one (thatis, at least one) of the grammatical object of the article, unlessotherwise made clear in the specific use of the article in only asingular sense.

The invention is to provide a method for treating neurodegenerationcaused by nerve compression syndrome or entrapment neuropathy comprisingadministering a fibrin glue mixture comprising human acidic fibroblastgrowth factor (aFGF) to a subject in need thereof.

The term “nerve compression syndrome” or “entrapment neuropathy” as usedherein refers to peripheral-nerve dysfunction that is due to localizedinterference of microvascular function and structural changes in thenerve or adjacent tissues. Nerve compression syndrome may be compressedby prolonged or repeated external force, such as sitting with one's armover the back of a chair (radial nerve), frequently resting one's elbowson a table (ulnar nerve), or an ill-fitting cast or brace on the leg(peroneal nerve). As the compression becomes more severe over time,focal demyelination occurs, followed by axonal damage, and finallyscarring. Moreover, some conditions cause nerves to be particularlysusceptible to compression. These include diabetes, in which the bloodsupply to the nerves is already compromised, rendering the nerve moresensitive to minor degrees of compression.

Common nerve compression syndromes include herniated intervertebraldisc, cervical nerve compression syndrome, thoracic outlet syndrome, andcarpal tunnel syndrome. Other nerve compression syndromes may includebut not limited to anterior interosseous syndrome, pronator teressyndrome, ligament of Struthers syndrome, cubital tunnel syndrome,Guyon's canal syndrome, radial nerve compression, posterior interosseousnerve entrapment, Wartenberg's syndrome, suprascapular nerve entrapment,peroneal nerve compression, tarsal tunnel syndrome, meralgiaparaesthetica, iliohypogastric nerve entrapment, obturator nerveentrapment, pudendal nerve entrapment, and abdominal cutaneous nerveentrapment syndrome.

The term “human aFGF” as used herein refers to a native human aFGF orany modified peptide from the native human aFGF. The modified peptidemay be obtained such as by one or more deletions, insertions orsubstitutions or combination thereof in the native human aFGF. In oneembodiment of the invention, the modified human aFGF is a peptidecomprising a native human aFGF shortened by a deletion of 20 amino acidsfrom N-terminal of the native human aFGF, and an addition of Alaninebefore the shortened native aFGF. For example, the human aFGF may be apeptide having the amino acid sequence of SEQ ID NO:1, which isdescribed in U.S. application Ser. No. 12/482,041, and herebyincorporated by reference herein in its entirety.

According to the invention, the human aFGF may be constituted into anyform suitable for the mode of administration selected. Preferably, thehuman aFGF is administered topically, intraneurally, or intramuscularly.According to the invention, the skilled person in the art may easilydetermine the administrative pathway and the dosage dependent on hisexperiments or the physiological status of the patents suffering nervecompression syndromes, i.e. aging, body weight, severity, or dosage. Inan embodiment of the invention, 0.1 mg/ml human aFGF were injected intothe left adductor hallucis muscles.

It is known that polypeptide or protein may easily degrade in vivo. Inorder to prolong the effect of human aFGF in a subject, human aFGF maybe included in any carriers or compositions for sustained release. Forexample, fibrin glue has been successfully used in a wide range ofsurgical fields such as skin graft fixation, nerve repair, cartilagereattachment and microvascular anastomoses. Researches also demonstratedsustained release of an active agent from fibrin glue for a longerperiod.

Thus, in one example of the present invention, human aFGF is containedin a fibrin glue mixture and administered to the subject suffering nervecompression syndrome or entrapment neuropathy. The concentration of theaFGF in the fibrin glue mixture is preferably about 0.01 mg/ml to 100mg/mi. In one embodiment of the invention, the fibrin glue mixturecontained 0.1 mg/ml aFGF when administered by adductor hallucis muscleinjection. In another embodiment of the invention, the fibrin gluemixture contained 0.04 mg/ml aFGF when administered by foramen magnuminjection. For those skilled in the art, the therapeutically effectiveamount, as well as dosage and frequency of administration, may easily bedetermined according to their knowledge and standard methodology ofmerely routine experimentation based on the present disclosure.

The fibrin glue mixture of the present invention referred to apharmaceutical composition contains human aFGF, fibrinogen, aprotininand divalent calcium ions, which may provide the effect of biocompatibility and sustained release. In one example of the invention,the divalent calcium ions can be any calcium ion sources, such as thoseprovided by addition of calcium chloride or calcium carbonate.

According to the invention, the fibrin glue mixture comprises 0.01-100mg/ml of human aFGF, and a fibrin glue containing 10-1000 mg/ml offibrinogen, 10-500 KIU/ml of aprotinin and 0.1-10 mM of calciumchloride. In one example of the present invention, in order to providebetter coagulation effect, the fibrin glue mixture may further comprise10-100 IU/ml thrombin.

The fibrin glue mixture of the present invention may be constituted intoany form suitable for the mode of administration selected. Preferably,the fibrin glue mixture is administered topically, intraneurally, orintramuscularly. According to the invention, the skilled person in theart may easily determine the administrative pathway dependent on hisexperiments or the physiological status of the patents suffering nervecompression syndromes, i.e. aging, body weight, severity, or dosage.

According to the invention, a neurodegenerative animal model wasdesigned to demonstrate the effect in promoting nerve fiber outgrowthand functional recovery. In one embodiment of the invention, the leftcervical dorsal root ganglion (DRG) was ligated with nylon suture 8-0.One week after ligation injury, total 0.6 μg aFGF in 6 μl normal salinewere injected into the left adductor hallucis muscles.

In another embodiment of the invention, one week after ligation injury,5 μl of fibrin glue mixture comprising 0.04 mg/ml (0.2 μg aFGF in 5 μlsaline), 10 mg/ml of fibrinogen, and 200 KIU/ml of aprotinin wasinjected directly into foramen magnum of the rats conducted the surgery.Five minutes after the first injection, 10 μl of supplementalcomposition comprising 0.45 mM of calcium chloride and 10 IU/ml thrombinwas injected into foramen magnum by the same injector.

The functional improvement was determined by a grasping power test. Asthe result thus obtained from the test on 2 and 3 weeks after thetreatment, the group administered with aFGF had a better effects onrecovering neural function in terms of a significantly high graspingpower ratio (injury side-L/normal side-R) than the sham control group(administered with saline only). Given the results, the presentinvention provides an unexpected treatment for recovery of neuralfunction.

As neural regeneration is time-consuming, continuous administration isrequired for repairing neural degeneration. In one example of theinvention, it was suggested to administer the subject the fibrin gluemixture comprising human aFGF once a month. Anyone skilled in the artcan determine the dose regiment according to the standard methods andcommon knowledge and the performance of the subject to be administered.

The present invention is more specifically explained by the followingexample. However, it should be noted that the present invention is notlimited to these examples in any manner.

EXAMPLE aFGF Rescues Neural Function in a Rat Model of CompressionNeuropathy

Animals

A total of twenty adult female Sprague-Dawley rats, aged between 8 and10 weeks, body weight 250-300 g were used. The animals were operated ona heating pad under general halothane anesthesia (1.0 liter/min to keepthe breathing rate at approximately 60/min). Rectal temperatures weremonitored and maintained during surgery. Bipolar electrocauterizationwas used to minimize bleeding. Antibiotics were injected subcutaneouslybefore the operation and one daily for 1 week afterward. After theoperation, animals were kept in ventilated, humidity- andtemperature-controlled rooms with a 12/12-h light/dark cycle.

Compression Surgery in Rats

The rats were placed in a prone position and their C4-C8 vertebrae wereexposed. Left C4 to C7 hemilaminectomies were carried out. While underthe microscope, the left C5 to C7 cervical roots were identified andfollowed after the overlying facets had been removed by drillingslightly more laterally in these segments. The dura was then carefullyopened and the C5 to C7 nerve roots were pulled tight and the C5, C6 andC7 section of left cervical dorsal root ganglion (DRG) were exposed andligated with the distal segment by nylon suture 8-0. After the surgery,these rats were divided into two experimental groups.

In Group A, the rats were divided into a sham control group (treatedwith saline, n=6), and aFGF group (treated with 0.6 μg aFGF in 6 μl,n=5). One week after the compression surgery, the rats were anesthetizedwith isoflurane and kept on the warm pad to maintain their bodytemperature, total 0.6 μg aFGF in 6 μl normal saline were slowlyinjected intramuscularly into adductor hallucis muscles (from 1^(st) to4^(th) pads, 1.5 μl/pad) in aFGF treatment group, while control groupwas injected with normal saline. The neural function recovery wasestimated by grasping power test (Table 1).

Alternatively in Group B, the rats were also divided into two groups: asham control group treated with saline (n=4), and aFGF treatment group(0.2 μg aFGF/in 5 μl saline, n=4). One week after the compressionsurgery, aFGF mixed with 5 μl of cocktail solution containing 1.5 mgfibrinogen (Beriplast P, Germany), apotinin solution (200 KIU/ml, 20 μl)and HBSS (Hank's balanced solution, 80 μl) was slowly injected into theforamen magnum in aFGF treatment group, while control group was injectedwith normal saline mixed with 5 μl of cocktail solution. Five minutesafter the injection of cocktail solution with aFGF or saline, 10 μl ofsecond solution containing 0.45 mM of calcium chloride and and 10 IU/mlthrombin was injected into the foramen magnum, and removed the injectorsa few seconds later. The neural function recovery was also estimated bygrasping power test (FIG. 1).

Grasping Power Test

The grasping power test was a modification of the method of Bertelli andMira (J. A. Bertelli et al., and J. C. Mira, Neurosci Methods 58:151-5,1995). For the assessment of grasping strength, a bar of wires wasconnected to an ordinary electronic balance. Both forepaws were tested,testing one forepaw at a time. The untested forepaw was temporarilyprevented from grasping by wrapping it with adhesive tape, and thetested forepaw was kept free. The rats were allowed to grasp the barwhile being lifted by the tail with increasing firmness until theyloosened their grip, and the grasping power was scored. The graspingpower index is the ratio of injured side/normal side (injuryside-L/normal side-R). Higher the score, better the functional recovery.

Table 1 demonstrated grasping result of Group A injected intramuscularlyinto adductor hallucis muscle. It was observed that the rats of aFGFtreatment group performed better grasping power than those in thecontrol group.

TABLE 1 Grasping Power Test Result of Group A (IntramuscularlyInjection) pre-treat- post-treat- post-treat- post-treat- Group Rat No.1 d 6 d 15 d 40 d aFGF A0055 0.647 0.743 — 0.830 A0056 0.714 0.366 0.9500.874 A0061 0.620 — 0.604 1.060 A0062 0.606 — 0.815 0.750 A0065 0.296 —— 0.945 AVERAGE 0.576 0.554 0.790 0.874 pre-treat- post-treat-post-treat- post-treat- Saline 2 d 5 d 14 d 44 d A0057 0.433 0.409 0.7210.382 A0058 0.686 0.540 0.433 0.895 A0059 0.353 0.448 — 0.579 A00600.479 1.078 0.715 1.077 A0063 0.581 — — 0.767 A0064 0.565 — — 1.234AVERAGE 0.516 0.619 0.623 0.733

The results of grasping power test for Group B was shown in FIG. 1. Twotailed Student's test was performed to examine statistic significancewith respect to the difference in grasping power between aFGF treatmentgroup and control group, or between pre-treatment and post-treatment ofaFGF. In the FIG. 1, the score of the rats administered aFGF wassignificantly higher than those administered saline on the 2 week and 3week after the treatment (*, p<0.05). On the other hand, the score ofthe rats administered aFGF after 2 week and 3 week were bothsignificantly higher than those before the treatment of aFGF (#,p<0.05). These studies indicated that administering a subject havingchronically/acutely compressed or mechanically injured nerves aFGF bythe foramen magnum, could rescue neural function. It is a breakthroughin the current filed of treating neural degeneration caused by nervecompression syndrome.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A method for treating neurodegeneration caused by nerve compressionsyndrome or entrapment neuropathy comprising administering a humanacidic fibroblast growth factor (aFGF) as the sole therapeutic agent toa subject in need thereof, wherein the human aFGF is mixed withfibrinogen, aprotinin and divalent calcium ions to form a fibrin gluemixture and then administered intrathecally or intramuscularly.
 2. Themethod according to claim 1, wherein the nerve compression syndrome orentrapment neuropathy is caused by a chronic compression, acutecompression, or a mechanical injury.
 3. The method according to claim 1,wherein the nerve compression or entrapment neuropathy is caused byherniated intervertebral disc, cervical nerve compression syndrome,thoracic outlet syndrome, or carpal tunnel syndrome.
 4. The methodaccording to claim 1, wherein the human aFGF comprises a deletion of thefirst 20 amino acids from N-terminus of the native human aFGF followedby an addition of Alanine at the N-terminus of the shortened nativeaFGF.
 5. The method according to claim 1, wherein the divalent calciumions are provided by the addition of calcium chloride or calciumcarbonate.
 6. The method according to claim 1, wherein the fibrin gluemixture comprises 0.01-100 mg/ml of human aFGF, 10-1000 mg/ml offibrinogen, 10-500 KIU/ml of aprotinin and 0.1-10 mM of calciumchloride.
 7. The method according to claim 1, wherein the fibrin gluemixture comprises 0.04 mg/ml human aFGF, 10 mg/ml of fibrinogen, 200KIU/ml of aprotinin and 0.45 mM of calcium chloride.
 8. The methodaccording to claim 1, wherein the human aFGF is administeredintrathecally.
 9. The method according to claim 1, wherein the humanaFGF is administered intramuscularly.
 10. The method according to claim8, wherein the human aFGF is administered through the foramen magnum.